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1.
Figure 7

Figure 7. Schematic representation of gADI functions during growth and encystation. From: ARGININE DEIMINASE PLAYS MULTIPLE REGULATORY ROLES IN THE BIOLOGY OF GIARDIA LAMBLIA.

A) During growth, the trophozoites acquire free arginine from the extracellular medium. Inside the cell, cytoplasmic gADI converts arginine into citrulline, with ATP production occurring at the final enzymatic step of the ADH pathway. B) gADI is released to the extracellular space when the trophozoites are in contact with human colon epithelial cells and compete with the host NOS for the free-arginine thereby reducing the production of NO. C) Under low exposure to specific antibodies, gADI acts as a peptidyl-arginine deiminase on the cytoplasmic tail of VSP inducing VSP switching. D) At the last step of encystation, gADI is translocated from the cytoplasm to the nuclei turning encystation specific genes off and ending the process.

Maria Carolina Touz, et al. J Cell Sci. ;121(Pt 17):2930-2938.
2.
Figure 5

Figure 5. gADI increases during Giardia differentiation. From: ARGININE DEIMINASE PLAYS MULTIPLE REGULATORY ROLES IN THE BIOLOGY OF GIARDIA LAMBLIA.

(A) IFA and confocal microscopy show the Giardia encystation process. CWP2 is synthesized and transported in ESVs (red) in encysting trophozoites (ET, arrowhead). At the end of the encystation process, CWP2 (red) is found in mature cyst walls (Cyst). Nuclei are stained with DAPI (blue). Scale bars represent 10 μm.
(B) Slot-blotting qualitatively shows gdh, gadi, and cwp2 gene expression at 0, 6, 24, and 48 hours of encystation in wild-type cells (WB/1267wt) and transgenic trophozoites (WB/1267-ADI+). The assay was performed in triplicate.
(C) Analysis of gdh, gadi, and cwp2 gene expression by RT-PCR and density quantification comparing both wild-type (WB/1267wt) and ADI-transgenic trophozoites (WB/1267-ADI+). Data represent the means ± s.d. for n=4 of two independent experiments.
(D) Western blotting using specific antibodies shows gADI, modified citrulline, VSP1267, and CWP2 protein expression in both wild-type (WB/1267wt) and ADI-transgenic trophozoites (WB/1267-ADI+). 10 μg of non-encysting (NT) and 24 h-encysting trophozoites (ET) were used for each sample. Mr is molecular mass expressed in kDa.

Maria Carolina Touz, et al. J Cell Sci. ;121(Pt 17):2930-2938.
3.
Figure 2

Figure 2. VSP citrullination is likely mediated by the PAD activity of gADI. From: ARGININE DEIMINASE PLAYS MULTIPLE REGULATORY ROLES IN THE BIOLOGY OF GIARDIA LAMBLIA.

(A) Confocal direct IFA was performed on permeabilized cells, showing a cytoplasmic distribution of gADI-HA (green) by using FITC-labeled anti-HA mAb, and its partial colocalization (yellow in merge) with VSP9B10 (red) underneath the plasma membrane of the transgenic trophozoite (top panels). The same result was obtained by using Alexa 488-anti-gADI in wild-type cells (bottom panels). Texas Red-9B10 mAb was used to visualize VSP9B10. Nuclei are stained with DAPI (blue). Scale bar represents 10 μm.
(B) Western blotting of Giardia homogenates expressing different VSPs reacted with anti-citrulline pAb (left pannel). The same filter membranes were stripped, cut, and reacted with 5C1, 9B10, and G10/4 mAbs against VSP1267, VSP9B10, and VSPH7, respectively (right panel), indicating that these VSPs are citrullinated. Mr is molecular mass expressed in kDa.
(C) Dot-blotting to detect citrullination of the H6-CRGKA peptide after incubation with the purified recombinant ADI-HA. A non-related purified enzyme ESCP-HA was used as a negative control. Dot-blotting to detect H6-CRGKA was performed by using anti-H6mAb.
(D) Specific citrullination of the CRGKA tail is shown (top panel). Western blotting using anti-citrulline pAb performed after immunoprecipitation with anti-HA mAb of H7-transgenic trophozoites. The presence of H7-HA, H7-tail, and ΔR-H7 after immunoprecipitation was analyzed by using anti-HA mAb labeled with alkaline phosphatase (bottom panel).

Maria Carolina Touz, et al. J Cell Sci. ;121(Pt 17):2930-2938.
4.
Figure 3

Figure 3. gADI participates in the control of cell death, probably by altering VSP switching. From: ARGININE DEIMINASE PLAYS MULTIPLE REGULATORY ROLES IN THE BIOLOGY OF GIARDIA LAMBLIA.

(A) Cytotoxicity assays of Giardia trophozoites WB/1267, GS/H7, and WB/1267 transfected with H7-HA or ΔH7 (X axis), is analyzed after 24 hours post-addition of anti-VSPH7 specific antibodies G10-4 and anti-GS-VSPs pAb by estimating the number of adherent viable parasites. Controls include cells without treatment (w/o mAb) and the use of a non-related mAb (8G8 mAb). Data represent the means ± s.d. for n=2 of three independent experiments.
(B) Progenies were analyzed by addition of goat anti-mouse FITC-conjugated antibody in IFA. Positive cells correspond to trophozoites expressing VSPH7. Nuclei are stained with DAPI (blue). DIC: Differential interference contrast. Scale bar represents 10 μm.
(C) VSP expression is established in WB/9B10 wild-type and WB/9B10-ADI+ transgenic trophozoites after a short time exposure to specific VSP9B10 mAb. Controls include no exposure to the mAb. Data represent the means ± s.d. for n=3 of two independent experiments.
(D) VSP9B10 citrullination is analyzed by Western blotting in WB/9B10 and WB/9B10-ADI+ trophozoites after a short time exposure to the anti-VSP9B10 mAb. The membrane labeled with anti-citrulline pAb, was stripped and reblotted with anti-VSP9B10 mAb. Similar amount of loaded VSP9B10 is observed. Molecular mass of VSP9B10 is indicated on the right.

Maria Carolina Touz, et al. J Cell Sci. ;121(Pt 17):2930-2938.
5.
Figure 4

Figure 4. Post-translational modifications of gADI. From: ARGININE DEIMINASE PLAYS MULTIPLE REGULATORY ROLES IN THE BIOLOGY OF GIARDIA LAMBLIA.

(A) Western blotting using anti-HA mAb shows gADI-HA in ADI-transgenic trophozoites. Multiple bands are also obtained using specific anti-gADI pAb in wild-type cells. The band of 64-66 kDa corresponding to the predicted protein sequences is observed in both cases (arrowhead) together with degradation products (gray arrows) including the low weight band (gray arrow with asterisk) found in the peptide pull-down (see Figure 1). Also, for both HA-tagged and native gADI, an increase in Mr from 64-66 kDa to 85 kDa band is shown (black arrow) suggesting that gADI undergoes posttranslational modification. Mr is molecular mass expressed in kDa.
(B) Western blot assays using anti-SUMO mAb recognize an ∼85 kDa band in both WB/1267 and GS/H7 Giardia clones. The same filter membrane was stripped and reblotted with gADI pAb, showing a perfect match with the higher band recognized by the gADI pAb. To confirm the lack of residual primary antibodies after stripping, only the secondary antibody was added to the stripped blots showing no signal (Control). Mr is molecular mass expressed in kDa.
(C) Western blotting using biotin conjugated anti-gADI pAb was performed to detect gADI bands after immunoprecipitation with anti-SUMO mAb. a) gADI in lysate before IPP, b) gADI after immunoprecipitation by using 0.1 μg of anti-SUMO mAb (arrow), c) gADI after immunoprecipitation by using 1 μg of anti-SUMO mAb (arrow), d) control using a non-related anti-HA mAb, and e) supernatant of sample c. Mr is molecular mass expressed in kDa.

Maria Carolina Touz, et al. J Cell Sci. ;121(Pt 17):2930-2938.
6.
Figure 6

Figure 6. During encystation, gADI is translocated to the nuclei and inhibits CWP expression. From: ARGININE DEIMINASE PLAYS MULTIPLE REGULATORY ROLES IN THE BIOLOGY OF GIARDIA LAMBLIA.

(A) Western blotting results after cytoplasm/nuclear fractionation assay using anti-gADI pAb at 24 and 48 hours post-encystation induction in wild-type (gADI) cells. (L): cell lysate previous fractionation, (C): cytoplasmic fraction, and (N) nuclear fraction (upper panel). Anti-VSP1267 mAb is used to detect cytoplasm/nuclear fraction contamination. Mr is molecular mass expressed in kDa (middle panel). IFA shows a representative time course gADI (red) distribution during encystation (ESVs in green, lower panel).
(B) Confocal microscopy and IFA using wild-type cells show that gADI (red) is translocated to the nuclei when the encysting cell is filled of ESVs (arrowheads). CWP1 is stained in green. Scale bar represents 10 μm.
(C) Top panels: Differential interference contrast (DIC) and DAPI staining show a representative image of an ADI-stable transgenic culture (Merge1). Middle panels: Direct IFA shows that trophozoites highly over-expressing gADI-HA in the nuclei (green) do not express CWP2 (red) during encystation (arrowheads in Merge2). Magnification 400 x. Bottom panel: Closer analysis of three ADI-transgenic cells demonstrated that the ones that show ADI-HA (green) on the nuclei (arrowheads) do not reveal CWP2 (red) expression after 24 h encystation while the low expressing ADI-HA does (arrow in Merge 3). Scale bar represents 10 μm.

Maria Carolina Touz, et al. J Cell Sci. ;121(Pt 17):2930-2938.
7.
Figure 1

Figure 1. Arginine deiminase (gADI) is associated with variant-specific surface proteins (VSPs) thorough their cytoplasmic tail. From: ARGININE DEIMINASE PLAYS MULTIPLE REGULATORY ROLES IN THE BIOLOGY OF GIARDIA LAMBLIA.

(A) Schematic representation of the pull-down assays (left). SDS-PAGE and Coomassie staining show an ∼85 kDa and an ∼13 kDa protein from both GS/H7 (GS) and WB/1267 (WB) isolates. Identification of these bands as gADI was performed by LC/MS-MS. Controls without peptide fail to pull down any protein (right).
(B) Two-hybrid interactions were detected by the ability of yeast cells (AH109) to grow on selective plates (TDO). In the upper left panel, the expression of VSPH7-BD, VSP1267-BD, 1267-tail-BD (VSP1267 lacking its cytoplasmic tail), and gADI-AD is revealed by the presence of white colonies in the –L/-T medium. Interaction of gADI-AD with both VSPH7-BD and VSP1267-BD is shown in the bottom left panel by the growth of yeast colonies in TDO plates. No interaction between gADI and 1267-tail-BD is observed. Controls of the methodology include ESCP-BD/gMuA-AD interaction (+) and emptyBD/gADI-AD vector (−). –L/-T: SD yeast medium lacking Leu and Trp. TDO stands for Triple Dropout Medium: SD/–His/–Leu/–Trp.
(C) Schematic representation of VSPH7 and transgenic VSPH7 proteins. VSPH7 ORF contains a signal peptide, an extracellular domain, a transmembrane domain (TM), and a cytoplasmic tail. H7-HA has a HA epitope sequence at the C-terminus, and the H7-tail possesses the HA epitope right after H7's TM. ΔR-H7 is VSPH7 containing a point mutation of the R residue of its cytoplasmic tail.
(D) H7-HA and ΔR-H7, but not H7-tail, co-immunoprecipitates with gADI. Antibody against gADI was used to immunoprecipitate comparable amounts of protein from WB transgenic cells. Western blotting of original lysate was stained with anti-HA mAb labeled with alkaline phosphatase (Lysate, bottom panel). UT stands for untransfected cells. Mr is molecular mass expressed in kDa.

Maria Carolina Touz, et al. J Cell Sci. ;121(Pt 17):2930-2938.

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